This invention relates to arrays, particularly polynucleotide arrays such as DNA arrays, which are useful in diagnostic, screening, gene expression analysis, and other applications.
Polynucleotide arrays (such as DNA or RNA arrays), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as xe2x80x9cfeaturesxe2x80x9d) are positioned at respective locations (xe2x80x9caddressesxe2x80x9d) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Biopolymer arrays can be fabricated by depositing previously obtained biopolymers onto a substrate, or by in situ synthesis methods. The in situ fabrication methods include those described in WO 98/41531 and the references cited therein. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides on a support by means of known chemistry. Typically these methods use a nucleoside reagent of the formula: 
in which:
A represents H or an optionally protected hydroxyl group;
B is a purine or pyrimidine base whose exocyclic amine functional group is optionally protected;
Q is a conventional protective group for the 5xe2x80x2xe2x80x94OH functional group;
x=0 or 1 provided:
a) when x=1:
R13 represents H and R14 represents a negatively charged oxygen atom; or
R13 is an oxygen atom and R14 represents either an oxygen atom or an oxygen atom carrying a protecting group; and
b) when x=0, R13 is an oxygen atom carrying a protecting group and R14 is either a hydrogen or a di-substituted amine group.
When x is equal to 1, R13 is an oxygen atom and R14 is an oxygen atom, the method is in this case the so-called phosphodiester method; when R14 is an oxygen atom carrying a protecting group, the method is in this case the so-called phosphotriester method.
When x is equal to 1, R13 is a hydrogen atom and R14 is a negatively charged oxygen atom, the method is known as the H-phosphonate method.
When x is equal to 0, R13 is an oxygen atom carrying a protecting group and R14 is either a halogen, the method is known as the phosphite method and, when R14 is a leaving group of the disubstituted amine type, the method is known as the phosphoramidite method.
The conventional sequence used to prepare an oligonucleotide using reagents of the type of formula (I), basically follows the following steps: (a) coupling a selected nucleoside through a phosphite linkage to a functionalized support in the first iteration, or a nucleoside bound to the substrate (i.e. the nucleoside-modified substrate) in subsequent iterations; (b) optionally, but preferably, blocking unreacted hydroxyl groups on the substrate bound nucleoside; (c) oxidizing the phosphite linkage of step (a) to form a phosphate linkage; and (d) removing the protecting group (xe2x80x9cdeprotectionxe2x80x9d) from the now substrate bound nucleoside coupled in step (a), to generate a reactive site for the next cycle of these steps. The functionalized support (in the first cycle) or deprotected coupled nucleoside (in subsequent cycles) provides a substrate bound moiety with a linking group for forming the phosphite linkage with a next nucleoside to be coupled in step (a). Final deprotection of nucleoside bases can be accomplished using alkaline conditions such as ammonium hydroxide, in a known manner.
The foregoing methods of preparing polynucleotides are described in detail, for example, in Caruthers, Science 230: 281-285, 1985; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al., Nature 310: 105-110, 1984; and in xe2x80x9cSynthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,869,643, EP 0294196, and elsewhere. The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach.
In the case of array fabrication, different monomers may be deposited at different addresses on the substrate during any one iteration so that the different features of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as the conventional oxidation and washing steps.
While each iteration of the foregoing sequence can have a very high yield (over 90%), there is still a small portion of the substrate bound moiety with unreacted linking groups (referenced together herein as xe2x80x9cfailed sequencesxe2x80x9d). It is known to cap such failed sequences to avoid the growth of undesired polynucleotide sequences from them. Capping compounds are described in the above mentioned references. A conventional capping compound is acetic anhydride which forms an acetate in conjunction with the hydroxy group of the substrate bound moiety. However, the yield of the capping reaction using acetic anhydride is relatively low. U.S. Pat. No. 4,816,571 suggests using a phosphite monoester capping reagent to form, along with the free hydroxy of the failed sequence, a phosphite triester blocking group. However, the present invention recognizes that in the fabrication of addressable arrays, use of such a capping reagent can leave some portions of the surface not carrying the desired polynucleotide sequences, with a different terminal group (a phosphite triester) than other portions since removal of the phosphite (de-capping) is relatively inefficient. This is particularly the case where an array is formed by a method which leaves spaces between the individual features (xe2x80x9cinterfeature spacesxe2x80x9d), such as deposition of droplets of reagents at the desired feature locations, and when capping is performed by exposing an entire functionalized substrate (such as by flooding) with the capping reagent. In such cases, some portions of the functionalized surface may be capped but not others. Due to such differences in interfeature surface composition (specifically, the functional groups left at the end the failed sequences or functionalizing group), background absorption of polynucleotides in a sample being tested onto interfeature areas may vary across the substrate, making identification of a features to which polynucleotides have bound, more difficult. This may be particularly the case where automated systems are used to detect such features, based on patterns observed on the array following exposure to a sample.
It is also known in the context of RNA hydrolysis generally, and in the context of preparing a xe2x80x9cuniversalxe2x80x9d solid support upon which oligonucleotides can be synthesized, that a xcex2-phosphotriester group (in relation to a an ester group) of a molecule used to link the growing oligonucleotide to a support, can be hydrolyzed so as to cleave the linker from the support and the phosphate from the linker to provide a 3xe2x80x2 hydroxy on the growing oligonucleotide. Such a scheme is disclosed in U.S. Pat. No. 5,681,945 and is illustrated in FIG. 1. Similarly, deBear et al. in Nucleosides and Nucleotides, 6(5), 821-830 (1987) also discloses preparation of a universal solid support involving the sequence illustrated in FIG. 2. Additionally, the reaction energy profile involved in reactions of the foregoing type, has been disclosed by Uchimaru et al., Biochemical and Biophysical Research Communications, Vol. 187, No. 3, 1523-1528 (1992). The foregoing references, and all other references cited in the present application, are incorporated herein by reference.
It would be desirable then, to provide an alternative method of capping failed sequences in polynucleotide formation. It would further be desirable to provide such a method which can be used in the fabrication of polynucleotide arrays and can provide failed sequences and interfeature areas with a functional group of the same type as provided by the functionalized surface.
The present invention then, provides a method of capping a hydroxy group of a moiety, comprising coupling the moiety to a xcex2-phosphor or xcex2-phosphite (such as a xcex2-phosphoramidyl), protected alcohol, so as to form the corresponding phosphate or phosphite between the hydroxy and phosphor or phosphite groups.
The invention may include the step of oxidizing any phosphorous ester linkage formed other than a phosphate (for example, phosphite) to the corresponding phosphate. The resulting compound (containing the phosphate group) may then be hydrolyzed to deprotect the protected alcohol and cleave the phosphate from the moiety so as to regenerate the hydroxy group of the moiety.
In another aspect, the present invention provides a method of capping and de-capping a hydroxy group of a moiety. In this aspect, the hydroxy group may be capped as described above, and de-capped by hydrolyzing the resulting compound to deprotect the protected alcohol and cleave the phosphate from the moiety so as to regenerate the hydroxy group of the moiety. The de-capping may be performed under suitable conditions, such as those described in deBear et al., cited above. For example, the hydrolysis may be performed under alkaline conditions.
In one aspect, the above method is applied to a method of synthesizing oligonucleotides on a substrate carrying substrate bound moieties each with a hydroxy group (such as a functionalized substrate surface or a hydroxy group of a substrate bound nucleotide). In this aspect, in a coupling step a first nucleoside carrying a phosphor or phosphite group is coupled to the hydroxy group of at least some of the substrate bound moieties in the usual manner. The first nucleoside has a protected hydroxy which can be deprotected under first deprotection conditions. In the case of phosphoramidite chemistry, this coupling step results in forming the corresponding phosphite between the hydroxy groups of the substrate bound moieties and the phosphoramidyl groups of the first phosphoramidite. At least some of the substrate bound moieties which failed to couple with the nucleoside phosphoramidite are capped in a capping step, by exposing them to a xcex2- or xcex3-phosphoramidyl, protected alcohol, in the manner described above, which protected alcohol can be deprotected under second deprotection conditions but not the first deprotection conditions. In the case of phosphoramidite chemistry, this forms the corresponding phosphite between the hydroxy of those substrate bound moieties, and phosphoramidyl group of the xcex2- or xcex3-phosphoramidyl, protected alcohol.
The oligonucleotide synthesis method of the present invention may include, following the foregoing capping, a deprotection step in which the substrate is exposed to the first deprotection conditions to deprotect the protected hydroxy of the coupled nucleoside in a manner already described. The sequence of the coupling, capping, de-capping steps, and de-protecting, may be repeated as often as required to form a desired polynucleotide, with the deprotected hydroxy of the coupled nucleoside from the deprotection step in one cycle of the steps, serving as the hydroxy group of substrate bound moieties in the next cycle. When all desired cycles are complete, the substrate may be exposed to the second deprotection conditions to de-cap failed sequences by hydrolysis in the manner already described, so as to regenerate the hydroxy group of the substrate bound moiety. It will be understood, of course, that there may be other optional steps provided in each cycle or at the end of all desired cycles. For example, an oxidation step may be provided to oxidize internucleoside phosphites to the more stable corresponding phosphates, and one or more washing steps may also be provided.
The present invention further includes, in another aspect, a method of fabricating an addressable array of polynucleotides on a substrate carrying substrate bound moieties each with a hydroxy group. This method includes, at each of multiple different substrate addresses, executing the above described olignonucleotide synthesis method of the present invention (particularly, including the described capping and de-capping steps). The phosphoramidites to be coupled at respective addresses may, for example, be deposited as droplets at those addresses, and wherein in the capping step at least inter-address areas (and preferably both address and inter-address areas) are exposed to the xcex2- or xcex3-phosphor or phosphite (for example, phosphoramidyl), protected alcohol.
The various aspects of the present invention can provide any one or more of a number of useful benefits. For example, an alternative method of capping failed sequences in polynucleotide formation is provided which does not require use of acetic anhydride. The method can be used in the fabrication of polynucleotide arrays and can provide failed sequences and interfeature areas with a functional group of the same type as provided by the functionalized surface.